The present proposal has to do with enterokinase and protein enterokinase inhibitors. Enterokinase is of interest because it is the initiator of protein digestion in vertebrates. It is also of interest because several enterokinase deficiencies have been discovered. Because it develops late in pregnancy the possibility is raised that premature infants may be deficient in enterokinase. Protein enterokinase inhibitors are of interest as antinutrients and as biochemicals that may be used to shut down digestive proteolysis in the duodenum if desirable. It is proposed that the observation that there are naturally occurring enterokinase inhibitors be pursued. One of the inhibitors, pancreatic trypsin inhibitor (Kunitz), is well characterized. The others are two peanut enterokinase inhibitors and Solanaceae (tomato/potato) enterokinase inhibitors. These require isolation and characterization. The latter would include amino terminal sequence analysis. Once homogeneous preparations of enterokinase inhibitors are on hand, it is proposed that the interaction of enterokinase with these inhibitors be studied for the purpose of determining whether they are likely to be potent enterokinase inhibitors if ingested. One approach to the problem will be thermodynamic and kinetic. Assay procedures suitable for studying proteases available in small quantities and kinetic procedures suitable for studying tightly binding inhibitors will be used. Another approach will have to do with the specificity of enterokinase inhibitors. The Laskowski reactive site cleavage procedure and chemical modification procedures will be used to determine enterokinase inhibiting sites on the protease inhibitors. By using protein protease inhibitors as probes, a better understanding of enterokinase will be generated. This includes an understanding of the relationship between the recently isolated membrane bound and soluble forms of the enzyme.